研究者詳細 - 島内　寿徳

Preparation of Nano- and Microparticles Obtained from Polymerization Reaction and Their Application to Surface Coating of Woody Materials 査読

Phase-separated cationic giant unilamellar vesicles as templates for the polymerization of tetraethyl orthosilicate (TEOS) 査読

Kohei Shiomi, Keita Hayashi, Haruyuki Ishii, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Sosaku Ichikawa

Biochimica et Biophysica Acta (BBA) - Biomembranes 1867 ( 2 ) 184403 - 184403 2025年2月

Toshinori Shimanouchi, Daichi Hirota, Masafumi Yoshida, Kazuma Yasuhara, Yukitaka Kimura

Applied Sciences 14 ( 23 ) 11326 - 11326 2024年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

A surface coating of polymer particles of different hydrophobicity and wide-ranged size is helpful for the surface modification of materials such as woody thin board (WTB) derived from biomass. A preparation method for polymer particles was, in this study, proposed using a capillary-type flow system. Under hydrothermal conditions, the refinement of dispersed oil droplets in water (O/W emulsions) and the polymerization reaction could be simultaneously advanced, and polymer particles of polystyrene (PS), polyvinyl alcohol (PVA), polymethyl methacrylate (PMMA), and poly-L-lactic acid (PLLA) with a particle size of about 100 nm could be synthesized. The coating of polymer particles gave an improved effect on the water repellency of WTBs due to the hydrophobicity of polymer particles and an alteration of surface roughness, and it also provided long-term stability (more than 6 years).

DOI： 10.3390/app142311326

Extraction of;ydroxymethylfurfural under hydrogen peroxide in liquid-liquid;slug flow of water;methylisobutylisoketone biphasic system;effect of hemin 査読

Toshinori Shimanouchi, Sota Sanagi, Satoko Fujioka, Koichi Terasaka, Yukitaka Kimura

Solvent Extr. Res. Dev. Japan 31 ( 2 ) 67 - 75 2024年4月

Classification of binding property of amyloid β to lipid membranes: Membranomic research using quartz crystal microbalance combined with the immobilization of lipid planar membranes. 査読国際誌

Toshinori Shimanouchi, Miki Iwamura, Yasuhiro Sano, Keita Hayashi, Minoru Noda, Yukitaka Kimura

Biochimica et biophysica acta. Proteins and proteomics 1872 ( 3 ) 140987 - 140987 2023年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

A biomembrane-related fibrillogenesis of Amyloid β from Alzheimer' disease (Aβ) is closely related to its accumulation behavior. A binding property of Aβ peptides from Alzheimer' disease to lipid membranes was then classified by a quartz crystal microbalance (QCM) method combined with an immobilization technique using thiol self-assembled membrane. The accumulated amounts of Aβ, Δfmax, was determined from the measurement of the maximal frequency reduction using QCM. The plots of Δfmax to Aβ concentration gave the slope and saturated value of Δfmax, (Δfmax)sat that are the parameters for binding property of Aβ to lipid membranes. Therefore, the Aβ-binding property on lipid membranes was classified by the slope and (Δfmax)sat. The plural lipid system was described as X + Y where X = L1, L1/L2, and L1/L2/L3. The slope and (Δfmax)sat values plotted as a function of mixing ratio of Y to X was classified on a basis of the lever principle (LP). The LP violation observed in both parameters resulted from the formation of the crevice or pothole, as Aβ-specific binding site, generated at the boundary between ld and lo phases. The LP violation observed only in the slope resulted from glycolipid-rich domain acting as Aβ-specific binding site. Furthermore, lipid planar membranes indicating strong LP violation favored strong fibrillogenesis. Especially, lipid planar membranes indicating the LP violation only in the slope induced lateral aggregated and spherulitic fibrillar aggregates. Thus, the classification of Aβ binding property on lipid membranes appeared to be related to the fibrillogenesis with a certain morphology.

DOI： 10.1016/j.bbapap.2023.140987

Amyloidogenic 60–71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects

Norihiro Namba, Takashi Ohgita, Hiroko Tamagaki-Asahina, Kazuchika Nishitsuji, Toshinori Shimanouchi, Takeshi Sato, Hiroyuki Saito

Scientific Reports 13 ( 1 ) 2023年12月

掲載種別：研究論文（学術雑誌）

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects.

DOI： 10.1038/s41598-023-45803-y

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein. 国際誌

Takashi Ohgita, Hiroki Kono, Izumi Morita, Hiroyuki Oyama, Toshinori Shimanouchi, Norihiro Kobayashi, Hiroyuki Saito

Scientific reports 13 ( 1 ) 10885 - 10885 2023年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

DOI： 10.1038/s41598-023-38070-4

Hydrothermal Preparation of Faceted Vesicles Made of Span 40 and Tween 40 and Their Characterization

Toshinori Shimanouchi, Yui Komori, Kazuki Toramoto, Keita Hayashi, Kazuma Yasuhara, Ho Sup Jung, Yukitaka Kimura

Applied Sciences (Switzerland) 13 ( 12 ) 2023年6月

掲載種別：研究論文（学術雑誌）

The Span 40 (sorbitan monooleate)/Tween 40 (polyoxyethylene sorbitan monolaurate) system gives faceted vesicles with angular surfaces, rather than spherical vesicles. Herein, a continuous and facile preparation method, based on the subcritical water-assisted emulsification and solvent diffusion, was presented to yield faceted vesicles with two major and minor axes (Type A) and vesicles closer to a polyhedron (Type B). Type A, rather than Type B, vesicles were likely to be formed. From the measurements concerning ζ-potential, membrane fluidity, and the polarization environment of the membranes, faceted vesicles could be obtained at 0.25 wt% of the surfactant concentration. The phase-separated behavior of Span 40 and Tween 40 within vesicle membranes could explain the structural feature of faceted vesicles and calcein leakage behavior. The significant advantage is that Type A vesicles would be utilized as alternative drug carriers for others with low encapsulation efficiency, although the present technical limitations cause difficulty in the selective formation of Type A and B vesicles and the selection of adequate solvent to accelerate the solvent diffusion step.

DOI： 10.3390/app13126893

Cholesterol as a Subsidiary Component of Sorbitan Surfactant-Based Aggregates: A Study of Formation, Hydrophobicity, and Estimation of Localization of Embedded Molecules. 国際誌

Keita Hayashi, Hikaru Ota, Haruna Sugimura, Toshinori Shimanouchi, Tomoyuki Iwasaki, Sakiko Fujita, Hidemi Nakamura, Hiroshi Umakoshi

The journal of physical chemistry. B 127 ( 10 ) 2214 - 2223 2023年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Aggregates of amphiphilic molecules can be used as drug carriers, for which the properties can be modified by mixing with other molecules such as cholesterol. It is important to understand the effects of such additives on the properties because they directly define the material functions. In this work, we investigated the effect of cholesterol on the formation and hydrophobicity of aggregates of sorbitan surfactants. As cholesterol changed its formation from micelles to vesicles, an increase in hydrophobicity was seen, particularly in the middle regions compared with the shallow and deep regions. We show that this gradual hydrophobicity is related to the localization of the embedded molecules. 4-Hydroxy-TEMPO and 4-carboxy-TEMPO were preferentially localized in the shallow region of the aggregates, whereas 4-PhCO2-TEMPO was preferentially localized in the deep region of the vesicle. The localization of molecules depends on their chemical structure. However, the localization of 4-PhCO2-TEMPO in micelles was not observed, despite the similar hydrophobicity in the hydrophobic region within the aggregates. The localization of embedded molecules was related to other properties, such as molecular mobility.

DOI： 10.1021/acs.jpcb.2c08153

Possible Role of Vesicles on Metallocatalytic Reduction Reaction of 5-Hydroxymethylfurfural to 2,5-Dimethylfuran 査読

Conversion of Glycerol to Lactic Acid by Using Platinum-supported Catalyst Combined with Phosphatidylcholine Vesicles

Toshinori Shimanouchi, Yuki Takahashi, Kazuma Yasuhara, Yukitaka Kimura

Chemistry Letters 52 ( 6 ) 426 - 429 2023年

Toshinori Shimanouchi, Yuki Takahashi, Keita Hayashi, Kazuma Yasuhara, Yukitaka Kimura

Compounds 2 ( 4 ) 321 - 333 2022年11月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

A reduction reaction of 5-hydroxymethylfurfural to 2,5-dimethylfuran (2,5-DMF) has been previously performed in an organic solvent under high-temperature conditions. For the relaxation of such reaction conditions, conventional palladium on carbon (Pd/C) was combined with vesicles composed of phospholipids or surfactants. Pd/C combined with 1,2-dioleoyl-sn-glycero-3-phosphocholine indicated a yield (25%) at 60 °C compared with Pd/C (17%). Vesicles at the liquid crystalline phase were advantageous for the reduction reaction of HMF. The yield of 2,5-DMF catalyzed by Pd/C combined with the vesicles depended on the lipid composition of the vesicles. It was clarified that the yield of 2,5-DMF could be controlled by the hydration property of the vesicles. Compared with conventional 2,5-DMF synthesis in an organic solvent, the use of vesicles made it possible to reduce the burden of using organic solvents in high-temperature conditions, although limitedly.

DOI： 10.3390/compounds2040027

Possible Role of Bent Structure of Methylated Lithocholic Acid on Artificial and Plasma Membranes

Tomoyuki Iwasaki, Nobuyuki Endo, Yuta Nakayama, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Keita Hayashi

Membranes 12 ( 10 ) 2022年10月

掲載種別：研究論文（学術雑誌）

Bile acids form micelles that are essential for the absorption of dietary lipids. However, excessive bile acid micelles can disrupt the plasma membrane by removing phospholipids, resulting in cell death. We hypothesized that the bent geometrical structure of the steroid scaffold of bile acids decreases the lipid order (similar to unsaturated phospholipids with cis double bonds), disrupting the plasma membrane. Here, lithocholic acid (LCA), a bile acid, was methylated to prevent micellization. Methylated lithocholic acid (Me-LCA) was mixed with a thin phase-separated lipid bilayer comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol). Me-LCA was not localized in the DPPC-rich rigid phase but localized in the DOPC-rich fluid phase, and excess Me-LCA did not affect the phase separation. Me-LCA is distributed in the plasma and organelle membranes. However, Me-LCA with bent structure did not affect the membrane properties, membrane fluidity, and hydrophobicity of liposomes composed of DOPC, DPPC, and Chol and also did not affect the proliferation of cells.

DOI： 10.3390/membranes12100997

Amyloid-β aggregates induced by β-cholesteryl glucose-embedded liposomes 査読

Toshinori Shimanouchi, Yasuhiro Sano, Kazuma Yasuhara, Yukitaka Kimura

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1870 ( 8 ) 140816 - 140816 2022年8月

Mechanisms of enhanced aggregation and fibril formation of Parkinson's disease-related variants of α-synuclein. 査読国際誌

Takashi Ohgita, Norihiro Namba, Hiroki Kono, Toshinori Shimanouchi, Hiroyuki Saito

Scientific reports 12 ( 1 ) 6770 - 6770 2022年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123-140 or 104-140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104-140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104-140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces.

DOI： 10.1038/s41598-022-10789-6

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates 査読国際誌

Naoyuki Iwahashi, Midori Ikezaki, Taro Nishikawa, Norihiro Namba, Takashi Ohgita, Hiroyuki Saito, Yoshito Ihara, Toshinori Shimanouchi, Kazuhiko Ino, Kenji Uchimura, Kazuchika Nishitsuji

Proceedings of the National Academy of Sciences of the United States of America 117 ( 52 ) 33225 - 33234 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

DOI： 10.1073/PNAS.2009931117

Fibril growth behavior of amyloid β on polymer-based planar membranes: Implications for the entanglement and hydration of polymers 査読

Shimanouchi, M Iwamura, S Deguchi, Y Kimura

Appl. Sci. 11 ( 10 ) 4408 2021年7月

Quantitative Determination of Relative Permittivity Based on the Fluorescence Property of Pyrene Derivatives: An Interpretation of Hydrophobicity in Self-Assembled Aggregates of Nonionic Amphiphiles. 査読国際誌

Keita Hayashi, Haruna Sugimura, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Hiroshi Umakoshi

The journal of physical chemistry. B 125 ( 23 ) 6192 - 6200 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Aggregates in aqueous solutions can embed hydrophobic molecules, and their interactions depend on the properties of the aggregates. The electric surface potential, molecular mobility, and gradual hydrophobicity are the properties that regulate the interactions, and it is essential to understand these to quantify the properties. Electric surface potential and molecular mobility are quantified using the zeta potential and NMR measurements. In this study, the quantification of gradual hydrophobicity within the aggregate based on the relative permittivity, also called the dielectric constant, has been estimated from fluorescence spectra of pyrene-dicarboxylic acid conjugates. The localization of the pyrene moiety was modified by conjugation with succinic acid, suberic acid, or dodecanedioic acid, and the conjugates were evaluated in the shallow, middle, and deep regions of the aggregates. Span and Tween surfactants have been employed to prepare these aggregates, because they form various kinds of aggregates such as micelles and vesicles. It was realized that the hydrophobicity gradually increased from the interface to the hydrophobic core. Alternatively, a comparison of hydrophobicity within the aggregates showed no remarkable difference. Moreover, the analyses suggested that there are a few water molecules in the deep region. These results support the idea of the localization of embedded molecules in aggregates.

DOI： 10.1021/acs.jpcb.1c00170

Microfluidic and hydrothermal preparation of vesicles using sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20). 査読国際誌

Toshinori Shimanouchi, Tetsuya Hayashi, Kazuki Toramoto, Saki Fukuma, Keita Hayashi, Kazuma Yasuhara, Yukitaka Kimura

Colloids and surfaces. B, Biointerfaces 205 111836 - 111836 2021年5月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

Here, we present a method for preparing vesicles by combining hydrothermal emulsification with solvent diffusion (SD). The sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20) system was used as the target lipid because these lipids are cheap and advantageous for the production scale. The water-in-oil (W/O) emulsion stabilized with lipids was formed under hydrothermal conditions (240 °C under 10 MPa), followed by mixing with water that included lipids to obtain a W/O-in-water (W/O/W) emulsion. The SD for the W/O/W emulsion as a subsequent process yielded vesicles. The optimal preparation conditions were 50:50 wt% Span 20/Tween 20 as a mixing ratio (final lipid concentration 12 mM), octanoic acid as an organic solvent, 240 °C for 4 min during the hydrothermal treatment, and 4 °C for 24 h in the SD process. The diameter of the vesicles obtained was at most 100 nm, which was comparable to that of the W/O/W emulsion before SD. This suggested that the W/O/W emulsion acted as a template for vesicle formation. The number density, diameter, and membrane properties of vesicles depend on the mixing ratio of the water/oil/lipid system. Specifically, the number density of vesicles was low relative to that of vesicles prepared by the conventional method.

DOI： 10.1016/j.colsurfb.2021.111836